Sporozoites of mammalian malaria parasites infect hepatocytes and therein the parasite develops as a liver stage. Liver stages are obligatory for infection, undergoing rapid growth and development, resulting in the formation of tens of thousands of red blood-cell infectious merozoites. Liver stages remain the least understood of all malaria life cycle stages and have only recently become amenable to systematic, detailed study. This is important as the liver stage is a promising target for new treatment and prevention modalities directed to halt parasite development prior to clinical manifestations of infection, either drugs or vaccines. Liver stages reside in a vacuolar compartment (parasitophorous vacuole, PV) that acts as a barrier to direct contact with the host hepatocyte cytoplasm. Consequently, any interaction of the parasite with its host cell takes place through this interface. We hypothesized in our initial grant that invasive sporozoites and liver stages export proteins that are targeted to the PV membrane (PVM) and that these proteins would be critical mediators of liver stage-hepatocyte interactions. Data generated from our studies provided convincing evidence in support of this hypothesis. We identified UIS3 and UIS4, small transmembrane proteins first expressed in the invasive organelles of sporozoites, later inserted into the PVM after invasion and continuously expressed during liver stage development. Independent deletion of UIS3 or UIS4 in rodent malaria parasites resulted in mutant parasites that are growth-arrested at the early liver stage, showing that UIS3 and UIS4 are each critical for successful liver stage development. We further determined that UIS3 directly interacts with host liver-fatty acid binding protein (L-FABP) and demonstrated that liver stage growth is directly proportional to L-FABP expression levels in hepatocytes. As such, this competitive renewal proposes to enhance our understanding for the functional role of parasite-host interface proteins to support Plasmodium liver stage growth and development. In our first aim we will map the interacting amino acid residues of UIS3 and L-FABP to determine if it is the direct interaction between these two proteins that is critical for parasite growth. We will also test L-FABP knockout mice for susceptibility to sporozoite infection and liver stage development. In our second aim we will identify hepatocyte proteins that interact with UIS4 and determine their importance for liver stage growth. The third aim will characterize recently identified novel putative liver stage-specific PV proteins and determine their significance for parasite growth in hepatocytes. In aims 1 and 2 we will expand our analyses of rodent parasite-host interactive proteins to human P. falciparum. The results of this project will present important data on mediators of liver stage-hepatocyte interactions that are essential for liver stage growth and survival. These interactions may be exploited for the design of novel strategies to prevent malaria infection. PUBLIC HEALTH RELEVANCE: The proposed competitive renewal research plan will determine the structural characteristics and functional role of parasitophorous vacuole membrane (PVM) proteins of the malaria parasite liver stage. We hypothesize that parasite liver stage PVM proteins interact with host hepatocyte proteins and that these interactions mediate obligatory resource acquisition essential for parasite growth and survival. Identifying major liver stage-host hepatocyte interactions and elucidating their functional significance will reveal fundamental principles of malaria parasite liver infection and provide new avenues for preventive drug design.